scholarly journals Selective STAT3 Degraders Dissect Peripheral T-Cell Lymphomas Vulnerabilities Empowering Personalized Regimens

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 865-865
Author(s):  
Giuseppina Astone ◽  
Luca Vincenzo Cappelli ◽  
William Chiu ◽  
Clarisse Kayembe ◽  
Rui Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
T Cell ◽  
Growth Inhibition ◽  
Research Funding ◽  
Granzyme B ◽  
Publicly Traded ◽  
T Cell Lymphomas ◽  
Peripheral T Cell Lymphomas

Abstract Introduction: Peripheral T-cell lymphomas (PTCLs) include heterogeneous entities of rare and aggressive neoplasms. The improved understanding of the biological/molecular mechanisms driving T-cell transformation and tumor maintenance has powerfully propelled new therapeutic programs. However, despite this progress, PTCLs remain an unmet medical need. Recurrent aberrations and the deregulated activation of distinct signaling pathways have been mapped and linked to selective subtypes. The JAK/STAT signaling pathway's deregulated activation plays a pathogenetic role in PTCL, including ALCL subtypes. STATs regulate the differentiation/phenotype, survival and cell-growth, metabolism, and drug resistance of T-cell lymphomas as well as host immunosuppressive microenvironments. Although many drugs' discovery programs were launched, a plethora of compounds has failed. Methods: We have discovered heterobifunctional molecules by an iterative medicinal chemistry SAR campaign that potently and selectively degrade STAT3 in a proteasome-dependent manner. Conventional PTCL cell lines and Patient Derived Tumor Xenograft (PDTX) and/or derived cell lines (PDTX-CL), carrying either WT- or mutated-STAT3, were exposed to increasing amounts (50nM⇒5µM) of STAT3-degraders. Proteins and mRNA transcripts (2⇒144hrs) were assessed by deep-proteomics and paired-end RNA sequencing, combined with WB/flow cytometry and qRT-PCR. Cell-titer-glo, cell titer blue, Annexin-V and S-cell cycle analyses were used as readouts. Chromatin accessibility, STAT3 DNA binding, 3D chromosomal architecture reorganization and 5-hmC profiling were assessed by ATACseq, CHIPseq and Hi-C and H3K27ac Hi-CHIP and mass-spectrometry. Drug testing/discovery combinations (96-well-plate) were performed using a semi-automated flow-cytometry. A battery of PTCL PDTX models were tested in pre-clinical trials. Results: Treatment of ALK+ ALCL (SU-DHL1) led to the rapid (~6hrs.) and profound down-regulation of STAT3 followed by the loss of canonical STAT3-regulated proteins (SOCS3, MYC, Granzyme B, GAS1, and IL2RA), without appreciable changes for other STAT family members (STAT1, STAT5b). In vitro, cytoplasmic, nuclear, and mitochondrial STAT3 downregulation was maintained up to 144 hrs. Loss of STAT3 in ALK+/- ALCL and BIA-ALCL cells was associated with major transcriptional changes (7116-10615 and 15114 DEGs in ALK- and ALK+ ALCL, respectively), underscoring public/shared as well as private time-dependent signatures. Main down-regulated pathways included JAK-STAT, MAPK, NF-kB, PI3K, TGFb, and TNFa. Comparison of STAT3 shRNA (ALK+ ALCL) and STAT3 degrader (ALK-/ALK+ ALCL) signatures demonstrated a substantial and concordant gene modulation (24hrs) among all models with the highest overlaps between ALK+ ALCL (Figure 3). To identify direct STAT3 gene targets, we analyzed CHIPseq peaks and predicted bindings sites, demonstrating that canonical genes, i.e., SOCS3, Granzyme B, GAS1, IL2RA, STAT3, and CD30, were significantly downregulated. Conversely, CD58, CD274, and MCH-I/II were upregulated at late time points. By mapping the STAT3 binding sites in ALK+ and ALK- ALCL, we have identified 1077 and 2763 STAT3 peaks within promoter/5'-/3'- and distant intergenic regions, corresponding to both coding and non-coding genes. Therapeutically, in vitro treatments led to cell cycle arrest and profound growth inhibition, and over time increased cell death of PTCL cells, including ALCL. Accordingly, growth inhibition of ALCL xenograft and PDTX tumors was also achieved (Figure 2). To identify drugs that could synergize withSTAT3-degrader activity, we tested a compound library (40) targeting pro-tumorigenic PTCL pathways as well as FDA-approved compounds. Ongoing studies are in progress. Conclusion: We have discovered selective STAT3 degraders which control PTCL growth. STAT3 degraders are powerful tools to define the STAT3 pathogenetic mechanisms and dissect genes/pathways to be targeted for T-cell lymphoma eradication. These data provide additional rationale for testing STAT3 degraders in the clinic for the treatment of aggressive malignancies including PTCL/ALCL. Figure 1 Figure 1. Disclosures Yang: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sharma: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Dey: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Karnik: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Elemento: Owkin: Consultancy, Other: Current equity holder; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Johnson and Johnson: Research Funding; Eli Lilly: Research Funding; Janssen: Research Funding; Champions Oncology: Consultancy; Freenome: Consultancy, Other: Current equity holder in a privately-held company; One Three Biotech: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding. Horwitz: Affimed: Research Funding; Aileron: Research Funding; ADC Therapeutics, Affimed, Aileron, Celgene, Daiichi Sankyo, Forty Seven, Inc., Kyowa Hakko Kirin, Millennium /Takeda, Seattle Genetics, Trillium Therapeutics, and Verastem/SecuraBio.: Consultancy, Research Funding; Acrotech Biopharma, Affimed, ADC Therapeutics, Astex, Merck, Portola Pharma, C4 Therapeutics, Celgene, Janssen, Kura Oncology, Kyowa Hakko Kirin, Myeloid Therapeutics, ONO Pharmaceuticals, Seattle Genetics, Shoreline Biosciences, Inc, Takeda, Trillium Th: Consultancy; Celgene: Research Funding; C4 Therapeutics: Consultancy; Crispr Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Forty Seven, Inc.: Research Funding; Kura Oncology: Consultancy; Kyowa Hakko Kirin: Consultancy, Research Funding; Millennium/Takeda: Research Funding; Myeloid Therapeutics: Consultancy; ONO Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy, Research Funding; Secura Bio: Consultancy; Shoreline Biosciences, Inc.: Consultancy; Takeda: Consultancy; Trillium Therapeutics: Consultancy, Research Funding; Tubulis: Consultancy; Verastem/Securabio: Research Funding. DeSavi: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liu: Kymera Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Growth Inhibition of Nasopharyngeal Carcinoma Cells Mediated by p53 Gene-Containing Nanolipid Composites

2020 ◽  
Vol 20 (10) ◽  
pp. 6026-6032
Author(s):  
Yongshan Cheng ◽  
Shanying Wu ◽  
Xinting Tie ◽  
Xiaodong Huang ◽  
Lihua Cui
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Nasopharyngeal Carcinoma ◽  
Growth Inhibition ◽  
Transfection Efficiency ◽  
P53 Gene ◽  
Theoretical Support ◽  
Novel Drugs ◽  
Cellular Apoptosis

To study the growth inhibition and cell cycle changes in nasopharyngeal carcinoma (CNE1) cells after transfection with p53 gene. A mixture of nano-liposomes and plasmid containing p53 was used for transfecting CNE1 cells. Cellular apoptosis was examined after transfection using the CCK-8 reagent method with flow cytometry. The results showed that a ratio of nanoliposome/p-ORF-GFP of 3.5:1 showed the highest transfection efficiency in CNE1 cells. The cells transfected with a mixture of composites in this proportion showed significant apoptosis of up to 50–70%. In addition, we observed that cell cycle changes-measured using flow cytometry-as well as cellular apoptosis were accelerated after administration of composites. The CCK-8 kit was used to determine the viability of nano-liposome-encapsulated p53 transfected cells. In vitro experiments showed that the combination significantly inhibited the growth of CNE1 cells with an inhibition rate of approximately 63.8%. Therefore, the nanocomposites have a significant effect on inhibiting the growth of CNE1 cells. Through the investigation of apoptosis and cell cycle changes in CNE1 cells we found that the nanoliposome-encapsulated p53 gene can inhibit growth in these cells, and might therefore serve as a novel treatment strategy for adjuvant treatment of nasopharyngeal carcinoma and ca also reduce incompatibility issues with radiotherapy and chemotherapy. This method can also provide technical and theoretical support for the development of novel drugs.

scholarly journals Granzyme B-expressing peripheral T-cell lymphomas: neoplastic equivalents of activated cytotoxic T cells with preference for mucosa- associated lymphoid tissue localization

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3785-3791 ◽  
Author(s):  
PC de Bruin ◽  
JA Kummer ◽  
P van der Valk ◽  
P van Heerde ◽  
PM Kluin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Lymphoid Tissue ◽  
Cytotoxic T Cells ◽  
Granzyme B ◽  
Clinical Behavior ◽  
T Cell Lymphomas ◽  
Hodgkin's Lymphomas ◽  
Peripheral T Cell Lymphomas

T-cell non-Hodgkin's lymphomas can be considered the neoplastic equivalents of immunologically functional, site-restricted T lymphocytes. Little is known about the occurrence and clinical behavior of T-cell lymphomas that are the neoplastic equivalents of different functional T-cell subsets. Here, we investigated the prevalence, preferential site, immunophenotype, and clinical behavior of the neoplastic equivalents of activated cytotoxic T cells (CTLs) in a group of 140 nodal and extranodal T-cell lymphomas. Activated CTLs were shown immunohistochemically with a monoclonal antibody against granzyme B, a major constituent of the cytotoxic granules of activated T cells. Granzyme B-positive T-cell lymphomas were mainly found in mucosa- associated lymphoid tissue (MALT; nose, 63% of the cases; gastrointestinal tract, 46%; and lung, 33%). Granzyme B-positive cases with primary localization in MALT were more often associated with angioinvasion (P = .005), necrosis (P = .002), and histologic characteristics of celiac disease in adjacent mucosa not involved with lymphoma. Eosinophilia was more often observed in granzyme B-negative cases (P = .03). Most cases belonged to the pleomorphic medium- and large-cell group of the Kiel classification. CD30 expression was more often found in granzyme B-positive lymphomas of MALT (P = .04), whereas CD56 expression was exclusively found in nasal granzyme B-positive lymphomas. Immunophenotypically, most of the cases should be considered as neoplastic equivalents of activated CTLs based on the presence of T- cell markers on tumor cells. In two cases of nasal lymphoma, tumor cells probably were the neoplastic counterparts of natural killer cells. The prognosis of the granzyme B-positive gastrointestinal T-cell lymphomas was poor but did not differ from granzyme B-negative gastrointestinal T-cell lymphomas. This indicates that, in peripheral T- cell lymphomas, site of origin is more important as a prognostic parameter than derivation of activated CTLs.

scholarly journals Utility of a Simple and Robust Flow Cytometry Assay for Rapid Clonality Testing in Mature Peripheral T-Cell Lymphomas

2019 ◽  
Vol 151 (5) ◽  
pp. 494-503 ◽  
Author(s):  
Natasha D Novikov ◽  
Gabriel K Griffin ◽  
Graham Dudley ◽  
Mai Drew ◽  
Vanesa Rojas-Rudilla ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Peripheral Blood ◽  
T Cell Clones ◽  
Flow Cytometric ◽  
T Cell Lymphomas ◽  
Cell Clones ◽  
T Cell Clonality ◽  
Cell Clonality ◽  
Peripheral T Cell Lymphomas

AbstractObjectivesFlow cytometry immunophenotyping is limited by poor resolution of T-cell clones. A newly described antibody was recently used to distinguish normal peripheral blood T cells from malignant T-cell clones. Here, we evaluate this antibody as a new diagnostic tool for detecting T-cell clonality in mature peripheral T-cell lymphomas.MethodsImmunostaining for the T-cell receptor β chain constant region 1 (TRBC1) along with routine T-cell markers was performed on 51 peripheral blood and two bone marrow samples submitted to the flow cytometry laboratory for suspected T-cell malignancy.ResultsTRBC immunophenotyping identified malignant T-cell clones with 97% sensitivity and 91% specificity. Findings correlated with molecular T-cell clonality testing. In cases with equivocal molecular results, TRBC1 immunophenotyping provided additional diagnostic information.ConclusionsTRBC1 flow cytometric immunophenotyping is a robust and inexpensive method for identifying T-cell clonality that could easily be incorporated into routine flow cytometric practice.

Selection and Characterization of Antibody Clones Are Critical for Accurate Flow Cytometry-Based Monitoring of CD123 in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1693-1693
Author(s):  
Nicole M. Cruz ◽  
Linda Lam ◽  
Mayumi Sugita ◽  
Roman Galetto ◽  
Agnès Gouble ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Surface Expression ◽  
T Cell Therapy ◽  
Transcript Levels ◽  
Car T Cell Therapy ◽  
Car T

Abstract Background: CD123, the trans-membrane alpha chain of the interleukin-3 receptor (IL-3RA) is overexpressed in acute myeloid leukemia (AML) and distinguishes leukemia stem cells from their normal counterparts. There are several novel therapeutics under development to target CD123 in AML, including CD123 fused to Diphtheria toxin, a recombinant chimeric anti-CD123 MoAb, CD3/CD123 bi-specific T cell engagers, and engineered T cells that express chimeric antigen receptors (CARs). Thus, accurate detection and quantification of CD123 is critical for newly diagnosed and relapsed patients, and to follow minimal residual disease for patients in remission. Our data suggest that the evaluation of CD123 by flow cytometry varies significantly with different antibody clones. Objective: To identify the most accurate flow cytometry method for evaluation of CD123 expression in patients with AML to evaluate CD123 targeting therapies. Methods: 51 AML patient samples and 7 normal cord blood or bone marrow samples were stained with five different commercially available monoclonal antibodies to detect CD123 (7G3, 6H6, 9F5, AC145 and FAB301P), as well as CD45 and CD5, for evaluation by multiparameter flow cytometry. CD123 gene expression was also compared between these primary AML samples and bone marrow samples from healthy donors. Cell surface expression (by percentage and MFI) was evaluated relative to transcriptional expression and sensitivity to known therapeutics (cytarabine, parthenolide, and HSP90 inhibitors). Results: We observed CD123 surface expression patterns varied between the antibody clones tested. For the 9F5 and 6H6 clones, 93% and 82% of the samples, respectively, showed >60% CD123+ cells whereas for the 7G3, FAB 301P and AC145 clones, 71 to 76% of the samples showed >60% positivity. Also, surface expression of CD123 using 7G3, AC 145 and FAB 301P did not correlate with transcript levels for IL3RA assessed using qPCR, while surface expression of CD123 using 9F5 and 6H6 did correlate with transcript levels of IL3RA, using both mean fluorescence intensity (MFI) and percentage. For example, the correlation between CD123 surface expression as measured by percentage and IL3RA transcripts was most significant using the 9F5 and 6H6 clone (R2=0.1084, p=0.0183, R2=0.1588, p=0.0038 respectively) whereas the correlation for 7G3 (R2=0.0004, p=0.8945), FAB301P (R2=0.0027, p=0.7151) and AC145 (R2=0.0392, p=0.1638) were not significant. Surface expression of CD123 evaluated with 7G3 antibody did not correlate with overall sensitivity to in vitro treatment with cytarabine (R2=0.03767, p= 0.6451). However, using the 9F5 antibody, we found that higher levels of surface CD123 were associated with resistance to cytarabine in vitro (R2= 0.5502, p= 0.0351). Differences were noted for other experimental therapeutics including parthenolide and PU-H71. Most importantly, when we tested the ability of a novel allogeneic anti-CD123 CAR T-cell therapy (UCART123) to eliminate CD123+ AML cells, we found that CD123 positivity as measured by the 7G3 clone was not predictive of sensitivity to UCART123 in vitro or in vivo AML patient derived xenotransplants. Conclusions: Several novel therapeutic modalities targeting CD123 in AML are under development, including allogeneic anti-CD123 CAR T-cell therapy. Accurate, quantitative assessment of CD123 expression is thus of utmost importance for patient selection in clinical trials as well as disease monitoring. We found discrepancies between antibody clones, and such discrepancies may alter patient selection and data interpretation regarding patient response to CD123 based therapies. For therapies targeting CD123, protocol design and antibody selection should be done considering the results in this study. Based on our findings we recommend 9F5 or 6H6 antibody clones as well as the utilization of qPCR along side flow cytometry for adequate detection. Flow cytometry findings should be reported as percent positive cells. If utilizing the 9F5 clone, samples with > 60% CD123+ should be considered positive for CD123. A comparison in a large cohort may be warranted to determine the impact of multiple CD123 measurements on disease outcome. Disclosures Galetto: Cellectis SA: Employment. Gouble:Cellectis: Employment. Smith:Cellectis SA: Employment. Roboz:Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy; Cellectis: Research Funding. Guzman:Cellectis: Research Funding.

scholarly journals Gabarap Loss Mediates Immune Escape in High Risk Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 891-891
Author(s):  
Annamaria Gulla ◽  
Eugenio Morelli ◽  
Mehmet K. Samur ◽  
Cirino Botta ◽  
Megan Johnstone ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Clinical Outcome ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Escape ◽  
Publicly Traded ◽  
Advisory Committees

Abstract Immune therapies including CAR T cells and bispecific T cell engagers are demonstrating remarkable efficacy in relapsed refractory myeloma (MM). In this context, we have recently shown that proteasome inhibitor bortezomib (BTZ) results in immunogenic cell death (ICD) and in a viral mimicry state in MM cells, allowing for immune recognition of tumor cells. Induction of a robust anti-MM immune response after BTZ was confirmed both in vitro and in vivo: treatment of 5TGM1 MM cells with BTZ induced tumor regression associated with memory immune response, confirmed by ELISPOT of mouse splenocytes. We have confirmed the obligate role of calreticulin (CALR) exposure in phagocytosis and the ICD process, since BTZ-induced ICD is impaired in CALR KO MM cells both in vitro and in vivo. We further showed that the therapeutic efficacy of BTZ in patients was correlated with ICD induction: BTZ-induced ICD signature was positively correlated with OS (p=0.01) in patients enrolled in the IFM/DFCI 2009 study. Together, these studies indicate that ICD is associated with long-term response after BTZ treatment. In this work, we reasoned that genomic or transcriptomic alterations associated with shorter survival of MM patients after BTZ treatment may impair activation of the ICD pathway. To this aim, we performed a transcriptomic analysis of purified CD138+ cells from 360 newly diagnosed, clinically-annotated MM patients enrolled in the IFM/DFCI 2009 study. By focusing on genes involved in the ICD process, we found that low levels of GABA Type A Receptor-Associated Protein (GABARAP) were associated with inferior clinical outcome (EFS, p=0.0055). GABARAP gene locus is located on chr17p13.1, a region deleted in high risk (HR) MM with unfavorable prognosis. Remarkably, we found that correlation of low GABARAP levels with shorter EFS was significant (p=0.018) even after excluding MM patients with del17p; and GABARAP is therefore an independent predictor of clinical outcome. GABARAP is a regulator of autophagy and vesicular trafficking, and a putative CALR binding partner. Interestingly, among a panel of MM cell lines (n=6), BTZ treatment failed to induce exposure of CALR and MM cell phagocytosis by DCs in KMS11 cells, which carry a monoallelic deletion of GABARAP. This effect was rescued by stable overexpression of GABARAP. Moreover, CRISPR/Cas9-mediated KO of GABARAP in 3 ICD-sensitive cell lines (AMO1, H929, 5TGM1) abrogated CALR exposure and ICD induction by BTZ. GABARAP add-back by stable overexpression in KO clones restored both CALR exposure and induction of ICD, confirming GABARAP on-target activity. Similarly, pre-treatment of GABARAP KO cells with recombinant CALR restored MM phagocytosis, further confirming that GABARAP impairs ICD via inhibition of CALR exposure. Based on these findings, we hypothesized that GABARAP loss may alter the ICD pathway via CALR trapping, resulting in the ICD resistant phenotype observed in GABARAP null and del17p cells. To this end, we explored the impact of GABARAP KO on the CALR protein interactome, in the presence or absence of BTZ. Importantly, GABARAP KO produced a significant increase of CALR binding to stanniocalcin 1 (STC1), a phagocytosis checkpoint that mediates the mitochondrial trapping of CALR, thereby minimizing its exposure upon ICD. Consistently, GABARAP KO also affected CALR interactome in BTZ-treated cells, which was significantly enriched in mitochondrial proteins. Importantly, co-IP experiments confirmed GABARAP interaction with STC1. These data indicate a molecular scenario whereby GABARAP interacts with STC1 to avoid STC1-mediated trapping of CALR, allowing for the induction of ICD after treatment with ICD inducers; on the other hand, this mechanism is compromised in GABARAP null or del17p cells, and the STC1-CALR complex remains trapped in the mitochondria, resulting in ICD resistance. To functionally validate our findings in the context of the immune microenvironment, we performed mass Cytometry after T cell co-culture with DCs primed by both WT and GABARAP KO AMO1 clones. And we confirmed that treatment of GABARAP KO clones with BTZ failed to activate an efficient T cell response. In conclusion, our work identifies a unique mechanism of immune escape which may contribute to the poor clinical outcome observed in del17p HR MM patients. It further suggests that novel therapies to restore GABARAP may allow for the induction of ICD and improved patient outcome in MM. Disclosures Bianchi: Jacob D. Fuchsberg Law Firm: Consultancy; MJH: Honoraria; Karyopharm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Richardson: AstraZeneca: Consultancy; Regeneron: Consultancy; Protocol Intelligence: Consultancy; Secura Bio: Consultancy; GlaxoSmithKline: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; Takeda: Consultancy, Research Funding; AbbVie: Consultancy; Karyopharm: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Abbvie: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Preclinical Activity of the Clinical Stage Protein Arginine Methyltransferase 5 (PRMT5) Inhibitor PRT543 in Splicing Mutant Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2597-2597
Author(s):  
Leya Y Schwartz ◽  
Gaurav S Choudhary ◽  
Nandini Ramachandra ◽  
Srabani Sahu ◽  
Shanisha Gordon ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Research Funding ◽  
Clinical Stage ◽  
Interleukin 1 ◽  
Splicing Factor ◽  
Myeloid Differentiation ◽  
Large Set ◽  
Publicly Traded ◽  
Exon 4

Abstract MDS and AML are generally incurable malignancies that need newer therapeutic options, as the disease-initiating stem cells are not eliminated by conventional therapies. Splicing factor mutations account for approximately 50% of mutations in MDS. Among those are SF3B1 and U2AF1 mutations, which are related to pathogenesis of disease by overactivation of oncogenic pathways, such as Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) signaling. Via activation of IRAK4 and other pathways, spliceosome mutations can lead to a block in differentiation and malignant proliferation. PRMT5 is an enzyme involved in spliceosome complex formation and fidelity and is over-expressed in patients with MDS/AML. The inhibition of PRMT5 may contribute to stem/progenitor cell differentiation rather than aberrant proliferation in an undifferentiated immature state. The objective of this study is to determine the activity of a clinical stage PRMT5 inhibitor, PRT543, in subtypes of MDS/AML using cell lines and primary samples. In preclinical studies PRT543 showed broad antitumor activity in vitro and in vivo (Bhagwat AACR 2020) and is currently under investigation in a Phase I clinical trial in patients with myeloid malignancies. We used a K562 cell line with CRISPR-introduced SF3B1 K700E mutation and isogenic control (K700K) in proliferation and myeloid differentiation assays with PRT543. The SF3B1 K700E mutant cells showed myeloid differentiation after treatment with the PRT543 PRMT5 inhibitor, as assessed by single cell colony assays and flow cytometry, while no substantial effects were observed in controls (K700K). We next evaluated PRMT5 expression in a large set of MDS CD34+ cells and observed substantial overexpression in SF3B1 mutant samples. Primary MDS/AML progenitors were cultured in methylcellulose colony forming unit (CFU) assays and treated with PRT543 at multiple concentrations versus vehicle controls. A majority of the SF3B1 patient samples showed a substantial increase in erythroid differentiation as assessed by colony formation and flow cytometry in the presence of 1nM and 5nM PRT543. In non-SF3B1 mutated patient samples, there was no clear difference in differentiation in the presence of PRT543. We next evaluated whether PRMT5 inhibition led to inhibition of oncogenic IRAK4 pathways. Retention of exon 4 of IRAK4 occurs in splicing mutant MDS and leads to production of an active long IRAK4 isoform. As measured by RNA-seq, PRMT5 inhibition led to decreased retention of exon 4 in IRAK4 transcripts. This decrease in the IRAK4 long form in response to PRT543 treatment was confirmed by immunoblotting, demonstrating reduction of this oncogenic signaling pathway. In summary, PRMT5 inhibition with PRT543 can release a differentiation block in MDS/AML, specifically in splicing mutant samples. PRMT5 inhibition decreases IRAK4-long isoform expression providing a potential mechanism for its activity in splicing factor mutant cases. Disclosures Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Heiser: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Starczynowski: kurome Inc: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; Stelexis: Current equity holder in publicly-traded company; Celgene: Consultancy; Acceleron: Consultancy; Novartis: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Curis: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; BMS: Research Funding.

Real-World Characteristics of Patients with Peripheral T-Cell Lymphoma Receiving Frontline Brentuximab Vedotin with Chemotherapy: A Retrospective Analysis with Propensity Score Matching

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 18-19
Author(s):  
John M. Burke ◽  
Nicholas Liu ◽  
Kristina Yu-Isenberg ◽  
Michelle A. Fanale ◽  
Andy Surinach ◽  
...  
Keyword(s):  
T Cell ◽  
Real World ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Primary Prophylaxis ◽  
Brentuximab Vedotin ◽  
Publicly Traded ◽  
Genetics Research ◽  
T Cell Lymphomas

Introduction: In the phase 3 ECHELON-2 study (NCT01777152), treatment with brentuximab vedotin (BV) + cyclophosphamide, doxorubicin, and prednisone (A+CHP) demonstrated significantly longer progression-free and overall survival compared with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) in the frontline (FL) treatment of patients with systemic anaplastic large cell lymphoma (sALCL) or other CD30-expressing peripheral T-cell lymphomas (PTCL). This study supported the November 2018 US FDA approval of A+CHP as FL therapy for adults with sALCL or other CD30-expressing PTCL. The current analysis describes patient characteristics, PTCL subtypes, and supportive care use of FL A+CHP and CHOP outside of the clinical trial setting in the US. Methods: Using medical and pharmacy claims data in the Symphony Health Solutions database, a retrospective cohort analysis of patients with PTCL treated with FL A+CHP or CHOP was conducted to compare treatment and utilization characteristics. Patients ≥18 years with 1 inpatient or 2 outpatient ICD-9/10 PTCL diagnosis codes, newly initiated on A+CHP or CHOP (index date) between November 2018 and January 2020, and with ≥6 months continuous activity before and ≥3 months after the index date were included. To adjust for confounding factors, a 1:1 propensity score matching analysis was performed based on age, gender, baseline comorbidities, geographic region and length of follow-up. Results: A total of 755 patients met inclusion criteria (335 A+CHP; 420 CHOP) with a median follow-up period of 10.1 and 10.6 months, respectively. In the unmatched cohorts, 61% were male, and median age at index was 62 and 69 years for A+CHP and CHOP, respectively. The prevalence of comorbidities based on the Charlson Comorbidity Index was similar between the cohorts; prevalent conditions included diabetes, chronic pulmonary disease, congestive heart failure, and liver disease (Table 1). PTCL subtypes treated with A+CHP included sALCL (54%), PTCL-not otherwise specified (NOS; 27%), and angioimmunoblastic T-cell lymphoma (AITL; 13%); subtypes treated with CHOP included PTCL-NOS (35%), adult T-cell leukemia/lymphoma (ATLL; 35%), and AITL (11%) (Table 2). After matching, the proportion of patients who received granulocyte-colony stimulating factor (G-CSF; A+CHP: 91%, CHOP: 86%, p=0.1) and the incidence rate of neutropenia (A+CHP: 45%, CHOP: 42%, p=0.4) during FL treatment for both study cohorts was similar. Of patients who received G-CSF, the majority received it as primary prophylaxis given within the first 5 days of FL treatment initiation (A+CHP: 89%, CHOP: 85%, p=0.2). The rate of subsequent therapy (ie, therapy change after FL), was similar between A+CHP and CHOP (18% vs 21%; p=0.3) and for the sALCL subtype (16% vs 26%, p=0.2). Of the A+CHP patients who received subsequent therapy, 32% were retreated with a BV-containing regimen and 19% of CHOP patients received a BV-containing regimen. Conclusions: In this real-world analysis, US patients with PTCL newly initiated on A+CHP or CHOP were older (67 vs 58 years) than those in ECHELON-2. There was a high comorbidity burden; over half of the patients in both cohorts had 1+ comorbidities, a potential reflection of the older population. As would be expected due to a high rate of CD30-positivity in the disease, A+CHP was more commonly used than CHOP in sALCL. In PTCL subtypes in which CD30 is more variably expressed, A+CHP and CHOP were used with similar frequencies. Although clinical trials in ATLL have demonstrated improved outcomes with more complex and intensive regimens than CHOP, CHOP remains commonly used in ATLL. A+CHP was also used in PTCL subtypes not included in ECHELON-2, such as NK/T cell lymphomas. G-CSF was used as primary prophylaxis in the large majority of patients in both cohorts. The use of a BV-containing regimen as subsequent therapy was more common in A+CHP vs CHOP, probably because the tumors of A+CHP patients were more likely to have expressed CD30. Confounding by unmeasured characteristics cannot be ruled out due to inherent limitations in claims data (eg, lack of disease stage, CD30 testing and response outcomes). Characteristics and management of this real-world population with PTCL differed from those in the ECHELON-2 trial, demonstrating the importance of retrospective studies to assess the impact of new regimens on clinical practice and to identify areas for further education of practitioners. Disclosures Burke: Seattle Genetics: Speakers Bureau; Gilead: Consultancy; Bristol Myers Squibb: Consultancy; Roche: Consultancy; Epizyme: Consultancy; Adaptive: Consultancy; Kura: Consultancy; Morphosys: Consultancy; Celgene: Consultancy; Adaptive Biotechnologies: Consultancy; Verastem: Consultancy; Astra Zeneca: Consultancy; Bayer: Consultancy; AbbVie: Consultancy. Liu:Seattle Genetics: Current Employment, Current equity holder in publicly-traded company. Yu-Isenberg:Seattle Genetics: Current Employment, Current equity holder in publicly-traded company. Fanale:Seattle Genetics: Current Employment, Current equity holder in publicly-traded company. Surinach:Seattle Genetics: Research Funding. Flores:Seattle Genetics: Research Funding. Lisano:Seattle Genetics: Current Employment, Current equity holder in publicly-traded company. Phillips:Beigene: Consultancy; AstraZeneca: Consultancy; Karyopharm: Consultancy; Bayer: Consultancy, Research Funding; Lymphoma Connect: Other; Incyte: Consultancy, Research Funding; Cardinal Health: Consultancy; University of Michigan: Current Employment; Pharmacyclics: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; BMS: Consultancy; Seattle Genetics: Consultancy.

scholarly journals Treatment-Free Intervals Mitigate T-Cell Exhaustion Induced By Continuous CD19xCD3-BiTE® Construct Stimulation in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 44-45
Author(s):  
Nora Zieger ◽  
Alyssa Nicholls ◽  
Jan Wulf ◽  
Gerulf Hänel ◽  
Maryam Kazerani Pasikhani ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Function ◽  
Granzyme B ◽  
T Cell Exhaustion ◽  
Specific Lysis ◽  
Cell Exhaustion ◽  
T Cell Function

The bispecific T-cell engager (BiTE®) blinatumomab is approved for treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and applied as continuous infusion over 28 days. The overall response rate to blinatumomab reported in clinical trials was 43 % and correlated to T-cell expansion (Zugmaier et al. 2015). In chronic viral infections, continuous antigen stimulation induces T-cell exhaustion, defined by phenotypic changes and functional impairment (Wherry 2011). Thus, we hypothesized that continuous BiTE® construct stimulation leads to T-cell exhaustion and that a treatment-free interval (TFI) reverses progressive T-cell dysfunction. To simulate continuous application of a BiTE® construct in vitro, T-cell long-term co-cultures were set up. Healthy donor T cells were stimulated in the presence of CD19+ OCI-Ly1 cells for 28 days with AMG 562, a half-life extended CD19 and CD3 specific BiTE® construct. T cells were harvested from the co-culture every 3-4 days between day 7 and 28 and assessed for markers of T-cell exhaustion: (1) AMG 562-mediated cytotoxicity of T cells was evaluated as specific lysis of CD19+ Ba/F3 target cells after 3 days, (2) T-cell expansion during the cytotoxicity assay was calculated as fold change (FC) of CD2+ counts, (3) Cytokine secretion of AMG 562-stimulated T cells was evaluated in co-culture supernatants by cytometric bead array (CBA) or after PMA/Ionomycine stimulation via intracellular cytokine staining (ICCS), (4) T-cell metabolic fitness was determined by Mito- and Glycolytic Stress Test using a Seahorse Analyzer, and (5) expression of the exhaustion-related transcription factor TOX was assessed by multiparameter flow cytometry. In order to assess the effect of a TFI on T-cell function, we cultured T cells and CD19+ OCI-Ly1 cells in the absence of AMG 562 from day 7-14 and 21-28 and compared their activity to T cells stimulated continuously with AMG 562. On day 7 of continuous (CONT) AMG 562 stimulation, we observed high cytotoxic and proliferative potential (% specific lysis=93±0.2, FC=2.9±0.2) as well as high IFN-g and TNF-a secretion analyzed by ICCS (% CD8+IFN-g+TNF-a+=23±6.7). However, cytotoxicity and proliferation decreased gradually until day 28 (% specific lysis=28±8.9; FC=0.6±0.1). CBA analysis confirmed decreasing secretion of IFN-g (day 3: 61113±12482, day 24: 3085±1351 pg/ml) and TNF-a (day 3: 1160±567, day 24: 43±7.6 pg/ml) as well as decreased IL-2 and granzyme B levels in culture supernatants. We furthermore observed highest mitochondrial fitness and basal glycolysis in T cells on day 7 of stimulation (basal OCR=2.2±0.6, maximal OCR=3.7±1.0, SRC=1.5±1.1 pmol/min/1000 cells, basal ECAR=2.0±0.4 mpH/min/1000 cells) which decreased until day 28 (basal OCR=0.4±0.2, maximal OCR=1.5±0.5, SRC=1.0±0.2 pmol/min/1000 cells, basal ECAR=0.5±0.2 mpH/min/1000 cells). In concordance, TOX increased during continuous stimulation (MFI ratio CD8+ day 7=6±0.8 to 12±0.8 on day 28). Strikingly, implementation of a TFI of 7 days led to superior cytotoxicity in T cells compared to continuously stimulated T cells (% specific lysis on day 14 CONT=34±4.2, TFI=99±2.2) and granzyme B production (CD8+; MFI ratio on day 14 CONT=124±11, TFI=303±34). Furthermore, increased proliferation during the cytotoxicity assay was observed in previously rested T cells (FC CONT=0.2±0.0, TFI=1.6±0.6). Although T cell function also decreased over time in TFI T cells, they maintained a strikingly higher cytotoxic potential (CONT=6±4.4, TFI=52±9.9) as well as higher granzyme B production (CONT=25±2, TFI=170±11) on day 28 compared to continuously stimulated T cells. In addition, TFI T cells showed increased IFN-g and TNF-a secretion after PMA/Ionomycine stimulation on day 28 (% CD8+IFN-g+TNF-a+ CONT=21±3.8, TFI=38±11.6). Our in vitro results demonstrate that continuous AMG 562 exposure negatively impacts T-cell function. Comprehensive analysis of T-cell activity in an array of functional assays suggests that continuous BiTE® construct exposure leads to T-cell exhaustion which can be mitigated through TFI. Currently, T cells from patients receiving blinatumomab are being analyzed to confirm the clinical relevance of our findings. Furthermore, RNA-Seq of continuously vs. intermittently AMG 562-exposed T cells will help us to understand underlying transcriptional mechanisms of BiTE® construct induced T-cell exhaustion. Disclosures Zieger: AMGEN Research Munich: Research Funding. Buecklein:Pfizer: Consultancy; Novartis: Research Funding; Celgene: Research Funding; Amgen: Consultancy; Gilead: Consultancy, Research Funding. Brauchle:AMGEN Inc.: Research Funding. Marcinek:AMGEN Research Munich: Research Funding. Kischel:AMGEN: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Subklewe:Gilead Sciences: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Morphosys: Research Funding; Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; Roche AG: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.

scholarly journals Venetoclax Enhances Anti-Leukemia Activity of CD123-Specific BiTE-Secreting T-Cells in AML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-13
Author(s):  
Hong Mu-Mosley ◽  
Lauren B Ostermann ◽  
Ran Zhao ◽  
Challice L. Bonifant ◽  
Stephen Gottschalk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Vehicle Control ◽  
Advisory Committees ◽  
Cellular Therapies

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p<0.001). In the Molm13 luciferase xenograft model, NSGS mice were randomized into 5 groups after AML engraftment was confirmed: 1) vehicle control, 2) Venetoclax (Ven) only, 3) CD123-ENG T-cells only, 4) Ven+CD123-ENG T-cells, 5) Ven+CD123-ENG T-cells/2-day-off Ven post T-cell infusion (Ven[2-day-off]+CD123-ENG). Venetoclax treatment (100 µg/kg daily via oral gavage) was started on day 4 post Molm13 injection, and on day 7, mice received one i.v. dose of CD123-ENG T-cells (5x106 cells/mouse). Venetoclax or CD123-ENG T-cell monotherapy reduced leukemia burden compared to the control group, and combinational treatments further inhibited leukemia progression as judged by BLI and circulating AML cells (%GFP+mCD45-/total live cells) by flow cytometry on day 15 post MOLM-13 injection: vehicle control: 19.6%; Ven+: 3.4%; CD123-ENG T-cells:1.2 %; Ven+CD123-ENG T-cells: 0.3%; Ven[2-day-off]+CD123-ENG T-cells (p<0.01 Ven+ or CD123-ENG T-cells versus control; p<0.001 Ven+CD123-ENG or Ven[2-day-off]+CD123-ENG T cells versus CD123-ENG T cells, n=5). The enhanced anti-AML activity of combining venetoclax and CD123-ENG T-cells translated into a significant survival benefit in comparison to single treatment alone (Fig. 2). However, while Ven+CD123-ENG and Ven[2-day-off]+CD123-ENG T-cell treated mice had a survival advantage, they had reduced circulating numbers of human CD3+ T cells on day 8 post T-cells infusion compared to mice that received CD123-ENG T-cells, indicative of potential adverse effect of venetoclax on T-cell survival in vivo. Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

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